Cleaved Serpin Refolds into the Relaxed State via a Stressed Conformer

Abstract

Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1 bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure. Under urea denaturing conditions, these cleaved proteins completely dissociated into the heavy (Gly 1 -Ala 352 ) and light (Ser 353 -Pro 385 ) chains. Upon refolding, the heavy chains of both proteins formed essentially the same initial burst refolding intermediates and then reassociated with the light chain counterparts. The reassociated intermediates both refolded into the native states with indistinguishable kinetics. The two refolded proteins, however, had a notable difference in thermostability. cl-OVA refolded into the stressed form with T m ‫؍‬ 68.4°C, whereas cl-R339T refolded into the relaxed form with T m ‫؍‬ 85.5°C. To determine whether cl-R339T refolds directly to the relaxed state or through the stressed state, conformational analyses by anion-exchange chromatography and fluorescence measurements were executed. The results showed that cl-R339T refolds first to the stressed conformation and then undergoes the loop insertion. This is the first demonstration that the P1-P1-cleaved serpin peptide capable of loop insertion refolds to the stressed conformation. This highlights that the stressed conformation of serpins is an inevitable intermediate state on the folding pathway to the relaxed structure. The serpins2 are believed to fold in vivo into a metastable "stressed form" as they undergo a conformational change to the thermostable "relaxed form" by insertion of the reactive center loop (RCL) between strands 3A and 5A (see Fig. 1) (1-3). The metastability of the native serpin is essential for the inhibition of serine proteinases, although this molecular flexibility also results in aberrant intermolecular linkage and polymerization that bring about conformational diseases such as dementia (4, 5), hepatocirrhosis (6), angioedema (7), and emphysema (8, 9). Therefore a central target of serpin studies is how the serpin polypeptide can fold into a metastable structure avoiding thermodynamically more stable conformations. Several serpin folding models have been suggested by various unfolding experiments, whereas refolding experiments from a fully unfolded state are limited (10 -14) because of serpin susceptibility to misfolding and aggregation.Ovalbumin is a major component of egg white proteins, and although it is not an apparent protease inhibitor its membership of the serpin family of protease inhibitors is clear from the close similarity of its primary and tertiary structure (15, 16). We have elucidated its refolding process from a fully denatured state as (10, 17, 18)

Cellular folding pathway of a metastable serpin

“…Constraining the gate itself may also be important, as conformational changes in this region have been implicated in pathological serpin polymerization (26), which can occur in the ER during serpin maturation. For the serpins α 1 -antitrypsin and ovalbumin, both of which lack C-terminal disulfide bonds, purified protein-folding studies have demonstrated the importance of the C-terminal region for proper folding and function (27)(28)(29)(30). These results highlight the importance of constraining conformationally labile regions early in serpin folding to efficiently reach the functionally required metastable structure.…”

Folding mechanism of the metastable serpin α 1 -antitrypsin

“…Electrostatic repulsion between K342 and K290 may promote polymerization by delaying the already slow insertion of strand 5A, which would be consistent with a loop sheet or RCL-strand 5A domain swap model for polymerization. Another possibility-suggested both by the study of Onda et al (12) and by that of Krishnan and Geirasch (6)-is that much of the molecule can adopt a native-like topology even before the formation of stable hydrogen bonds. In this case, positioning of β-strand 5A across the front of sheet B (but not its stable incorporation into sheet A) may be required for the subsequent folding of the C-terminal residues to proceed (see note).…”

“…The importance of proper formation of the B-C barrel in serpin folding is also highlighted by a study of an ovalbumin mutant that spontaneously adopts the stable loop inserted structure (12). When a fragment consisting of the first 352 residues was diluted from 8 M urea, it adopted a structure with the properties of the metastable state, including an exposed RCL.…”

The Mechanism of Fibril Formation of a Non-inhibitory Serpin Ovalbumin Revealed by the Identification of Amyloidogenic Core Regions

“…Limited Proteolysis-The core region involved in the fibril was mapped by identifying the local region that is not susceptible to proteolytic cleavage as reported previously (22,40,43). A 2 mg/ml solution of OVA was heat-treated at 80°C for 1 h and then digested with trypsin for 30 min at 37°C.…”

“…Proteolytic cleavage generally occurs at flexible regions of a polypeptide chain, and the structured regions of the amyloid fibril correspond to the most highly protease-resistant region. In this study, the protease-resistant region of the OVA fibril was assessed using trypsin, which is a particularly appropriate protease for these studies because the native conformation of OVA is highly resistant to trypsin but not in its non-native conformation (22,40,43). The heat-induced aggregates of intact OVA and SH-OVA were treated with trypsin, and the cleavage products were separated by 15% SDS-PAGE.…”